Fig. 4. mls-2 encodes a homeodomain protein of the HMX family. (A) mls-2 gene structure and constructs. The upper diagram represents the six exons of the mls-2 genomic sequence, with the molecular lesions in cc615 and tm252 indicated. Shaded areas represent the homeodomain. The lower diagram depicts various fusion constructs used in this work (see Materials and methods). Both pYJ59 and pYJ62 were able to rescue the mls-2(cc615) mutant phenotypes. (B) A Clustal W sequence alignment of MLS-2, other HMX (Nkx5) family homeodomain proteins and the C. elegans Nkx2.5 homolog CEH-22. Only residues within the homeodomain (residues 1-60) and two additional domains (in dashed boxes) immediately after the homeodomain were aligned. The proteins share no similarity outside this compared region. Identical amino acids are in white letters against black background. Similar amino acids are in black letters against gray background. The alignment is shown for CeMLS-2 (this work), C. briggsae CBG14538(WormBase), SOHo1 (chicken) (Deitcher et al., 1994), SpHmx (sea urchin) (Martinez and Davidson, 1997), TgHbox5 (sea urchin) (Wang et al., 1990), GH6 (chicken) (Stadler et al., 1994), DHmx (Drosophila) (Wang et al., 2000), murine Hmx3 (Nkx-5.1) (Bober et al., 1994), murine Hmx2 (Nkx-5.2) (Bober et al., 1994), human HMX2 (Wang et al., 2000), murine Hmx1 (Yoshiura et al., 1998), human HMX1 (H6) (Stadler et al., 1992) and CEH-22 (Okkema and Fire, 1994). (C) A phylogenetic tree of the HMX family generated using the neighbor-joining method of Clustal W. The tree is rooted with CEH-22 in TreeView version 1.6.6 (Page, 1996). Only the homeodomain and the two additional conserved domains immediately after the homeodomain (as shown in B) were used in the analysis.